The present invention relates to novel processes and synthetic intermediates for the preparation of xcex1-difluoromethyl ornithine.
Eflornithine or xcex1-difluoromethylornithine (DFMO) has recently been approved in the United States in a topical cream for removing unwanted facial hair. Efficient, scaleable syntheses of DFMO are therefor useful to provide manufacturing
A preparation of DFMO have been described previously in U.S. Pat. No. 4,309,442 from ornithine. The relatively high cost of the starting material, ornithine, and the use of desirable reagents including flammable reagents, however, makes the route less attractive for commercial manufacture.
An alternative preparation of DFMO was described in Swiss patent CH 672 124 from starting materials including malonic acid esters and acrylonitrile. The process is burdened by the use of a Hoffman type reaction which is a potential run-away reaction.
From a manufacturing standpoint it would be advantageous to have a process for the synthesis of DFMO that utilizes readily available and inexpensive starting materials. Processes for DFMO that avoid potentially explosive reaction conditions are also highly desirable. In addition, processes for DFMO production that would avoid the use of halogenated solvents, which require costly waste disposal protocols and emissions monitoring are also preferable.
In one embodiment, the invention relates to processes for the preparation of DFMO, having the formula 
The processes include the step of selectively reducing a nitrile moiety of a compound of the formula 
wherein R1 is linear or branched C1 to C4 alkyl and Z is (i) xe2x80x94NH2 or (ii) a protected amino moiety selected from the group consisting of 
wherein R2 is hydrogen, linear or branched C1 to C4 alkyl or aryl, and 
wherein R4 is linear or branched C1 to C4 alkyl, alkoxy or aryl.
In another or the same step, the 
moiety, if present, is hydrolyzed, producing as a result of the reduction step, or the reduction followed by hydrolysis steps, a compound of one of the following formulas 
In another step the ester and amide (including the lactam) moieties of formulas 7, 9, or 10 are hydrolyzed to give the compound of formula 1.
In another aspect, the invention relates to intermediates useful in the preparation of DFMO. The intermediates include compounds of the formula 
wherein R5 is: 
wherein R2 is hydrogen, linear or branched C1 to C4 alkyl or aryl; 
wherein R4 is linear or branched C1 to C4 alkyl, alkoxy or aryl. Preferred intermediates include: ethyl 2-benzylideneamino-2-difluoromethyl-4-cyanobutanoate, ethyl 2-(diphenylmethylene)amino-2-difluoromethyl-4-cyanobutanoate, ethyl 2-amino-2-difluoromethyl-4-cyanobutanoate, or ethyl 2-acetylamino-2-difluoromethyl-4-cyanobutanoate, or salts thereof.
In accordance with the present invention, novel processes and intermediates for the preparation of difluoromethyl ornithine (DFMO or the compound of formula 1) are provided. The processes of the invention have been developed from readily available and inexpensive starting materials. Furthermore, the processes provide high yields of DFMO, simplify isolation and purification steps, and minimize the use of halogenated solvents.
In one embodiment of the invention an alkyl glycine ester of the formula 2 serves as a convenient starting material for a short synthesis of an alkyl 2-difluoromethyl-4-cyanobutanoate intermediate (compound of the formula 5) wherein R1 is C1 to C4 linear or branched alkyl and R2 is hydrogen, C1 to C4 linear or branched alkyl, or aryl. Compound of the formula 5 can then be converted by a number of processes to DFMO. 
The compound of formula 3 can be obtained from the glycine ester of the formula H2NCH2CO2R1, (formula 2) wherein R1 is C1 to C4 alkyl. Preferably the alkyl group is methyl, ethyl, or t-butyl. Glycine ethyl ester, for example, is readily available from a number of commercial vendors as its hydrochloride salt. The compound of the formula 3 can be formed by treatment of the glycine ester of the formula 1 with an aryl aldehyde or ketone of the formula PhC(O)R2, wherein R2 is hydrogen, C1 to C4 alkyl or aryl (Scheme 1). A dehydrating agent such as magnesium sulfate or sodium sulfate can optionally be used to remove the water generated in the reaction. If the glycine ester of the formula 2 is provided as an acid addition salt, a tertiary amine base, e.g., triethylamine (TEA), tributylamine (TBA) or N,N-diisopropylethylamine, can be included in the reaction mixture to generate the neutral form of the ester.
While conventional methods for the preparation of Schiff""s base derivatives of glycine alkyl ester utilize halogenated reaction solvents such as dichloromethane, applicants have found that the reaction for the preparation of aldimine type intermediates (R2=H), can be advantageously carried out in acetonitrile at temperatures of about 10 to 35xc2x0 C., preferably at about 20 to 25xc2x0 C. The use of acetonitrile as a reaction solvent simplifies reaction work-up procedures and processing. The magnesium sulfate and tertiary amine base-acid addition salt (if used) can be simply removed by, for example, filtration, and the filtrate used directly in the next synthetic step, where acetonitrile also serves as the reaction solvent.
These reaction conditions can provide high yields and conversions, preferably  greater than 98% for both yield and conversion, of compound of the formula 3.
In embodiments of the process where the amino group is protected as a ketone imine (i.e., R2=C1 to C4 alkyl or aryl) the condensation reaction can also be accomplished using an aprotic solvent, e.g., xylene or toluene (preferably toluene), and catalytic amount of a Lewis acid, e.g., boron trifluoride etherate, triphenyl boron, zinc chloride, aluminum chloride, and the like. The condensation reaction can include the use of a Dean Stark trap and/or the use of other such dehydrating techniques known to those of ordinary skill to hasten the reaction rate by removing the formed water effectively.
The alkyl 4-cyanobutanoate of the formula 4 is, in one embodiment, obtained from the compound of the formula 3 by a Michael reaction. For example, compound of the formula 3 is treated with acrylonitrile, a base such as potassium carbonate and a phase transfer catalyst (PTC), such as triethylbenzylammonium chloride, tetrabutylammonium chloride, tetraethylammonium chloride, or trimethylbenzylammonium chloride at temperatures of from about 10 to about 45xc2x0 C., preferably from about 20 to 35xc2x0 C. Methods for the phase transfer catalyzed Michael addition of xcex1-amino acids wherein the xcex1-amino groups are protected as benzaldimines can be found in Yaozhong et al., Tetrahedron 1988, 44, 5343-5353.
The compound of formula 4 is then alkylated using a strong base and a halodifluoromethane alkylating reagent to form the compound of formula 5. Suitable strong bases include those that are effective in deprotonating the compound of formula 4 at the position xcex1 to the carboxylate. Examples of strong bases include alkali metal alkoxides of the formula MOR3 wherein M is Na, Li or K and R3 is C1 to C4 linear or branched alkyl; alkali metal hydrides, or alkali metal amide (e.g., sodium amide, sodium bistrimethylsilylamides). Preferably the alkoxide base is either a sodium or potassium alkoxide, more preferably a sodium alkoxide, such as sodium ethoxide or sodium t-butoxide. Preferably, a slight molar excess of base is used in the reaction such as from about 1.6 to 2.0 equivalents.
The alkylation reaction is carried out, for example, by deprotonation at a temperature of from about xe2x88x9235 to about 25xc2x0 C. Once the xcex1-anion has been generated, the alkylating reagent is introduced and the temperature of the reaction can be, for example, from about xe2x88x925 to about 20xc2x0 C. (for R2=aryl). Useful halodifluoromethane alkylating reagents include difluoroiodomethane, chlorodifluoromethane, or bromodifluoromethane. Preferably the halodifluoromethyl alkylating reagent is chlorodifluoromethane. Typically an excess of the halodifluoromethyl alkylating reagent is used in the reaction such as from about 1.05 to about 2.0 molar equivalents. In instances where the alkylation reaction is run in a pressure vessel, smaller amounts of the halodifluoromethyl alkylating agent are used.
The alkylation reaction is carried out in suitable aprotic solvents such as dimethylformamide, acetonitrile, N-methylpyrrolidone, dimethylsulfoxide, or an ether such as tetrahydrofuran, 2-methyltetrahydrofuran, methyl t-butyl ether, diethyl ether, dioxane, or mixtures thereof. Preferably the solvent used in this alkylation reaction is an ether, preferably tetrahydrofuran or tetrahydrofuran/acetonitrile.
In Process A (Scheme 2, Route I) for the synthesis of DFMO, the synthetic steps include: hydrolysis of the Schiff""s base protecting group, reduction of the nitrile moiety and hydrolysis of the alkyl ester moiety. The Schiff""s base protecting group of the compound of the formula 5 is hydrolyzed by treatment with an aqueous acid using conditions well known in the art, to provide the compound of formula 6. Suitable acids include mineral acids, toluenesulfonic acid, methanesulfonic acid, trifluoroacetic acid, and the like. The reaction is conveniently carried out in a mixture of the aqueous acid and an organic solvent. For example, a mixture of methyl t-butyl ether and 4 N HCl is stirred at ambient temperature to effect hydrolysis of the Schiff""s base. After the reaction mixture is made basic with hydroxide solution, the compound of formula 6 isolated in neutral form and used directly without further purification in the next synthetic step. 
The compound of the formula 6 is then converted to the diamino compound of formula 7 by reduction of the nitrile moiety. Any reduction procedure effective to selectively reduce the nitrile moiety to the amine with minimal competing ester reduction can be used. For example, heterogeneous transition metal catalysts are effective catalysts for the hydrogenation of the nitrile moiety. Typically an acid such as hydrochloric acid is added to the reaction mixture. The transition metal catalysts include, for example, palladium on carbon, platinum on carbon, and platinum oxide. Preferably the catalyst used in the reduction is 5-10% platinum on carbon.
The amount of hydrochloric acid typically used in the reaction is 1 to 5 equivalents, more preferably 3 to 4 equivalents. The reaction solvent for the hydrogenation can be an alcohol, preferably ethanol or an ether, preferably t-butyl methyl ether. The reaction is carried out in a suitable corrosive resistant reaction vessel such as a Hastelloy bomb vessel with hydrogen at a pressure of, for example, from about 80 to about 120 psi. The hydrogenation is typically run at temperatures from about 25 to about 40xc2x0 C., preferably about 25 to 30xc2x0 C.
DFMO can be obtained by hydrolysis of the alkyl ester moiety of the diamino compound of the formula 7. In one embodiment, the alkyl ester moiety can be hydrolyzed using aqueous basic conditions well known to those of ordinary skill in the art. Alternatively, the alkyl ester moiety can be hydrolyzed using acidic conditions. Suitable acids for the hydrolysis reaction include mineral acids or toluene sulfonic acid. Preferably the hydrolysis is effected using an excess of mineral acid, for example 12 N HCl at reflux. In the instance where R1 is a t-butyl ester, the t-butyl ester can also be hydrolyzed by milder acidic hydrolysis methods well known in the art, such as treatment with formic acid or trifluoroacetic acid.
DFMO can be conveniently isolated as its monohydrochloride monohydrate salt. For example, a solution of from about 9 to about 13% by weight DFMO in about a 1 to 3-3.5 mixture of aqueous hydrochloric acid 12 N and alcohol, preferably ethanol, is provided with a pH of less than 0.5. The resulting solution can be treated with sufficient triethylamine to effect a pH of about 4 to form a slurry containing precipitated DFMO dihydrochloride. The precipitated DFMO hydrochloride monohydrate is recovered by methods well known to those of ordinary skill in the art including filtration and centrifugation. The crude DFMO recovered can be further purified by recrystallization from suitable recrystallizing solvents such as ethanol/water. Preferably the purity of the DFMO is at least 98%, more preferably at least 99% pure.
In alternative embodiments of Process A, metal hydride reagents can be used to effect the selective reduction of the nitrile moiety. These hydride reagents include NaBH3(O2CCF3) and other such modified borohydride and aluminum hydride reagents that selectively reduce the nitrile moiety in the presence of a carboxylic ester moiety.
In a closely related embodiment of this metal hydride process, the ester moiety of the amino compound of formula 6 is hydrolyzed before reducing the nitrile group with a metal hydride. For example the alkyl ester of the amino compound of formula 6 is saponified to give a carboxylate salt. The nitrile is then selectively reduced to the amine by treatment with hydride reagents such as NaBH3(O2CCF3), and other such modified borohydride and aluminum hydride reagents that selectively reduce the nitrile moiety in the presence of a carboxylic acid or acid salt.
In another embodiment of Process A (Scheme 2, Route II), the compound of formula 5 is directly hydrogenated to form the diamino compound of formula 7 using a heterogeneous transition metal catalyst, e.g., platinum on carbon, using hydrochloric acid and a solvent such as ethanol.
In Process B, the compound of the formula 6 is converted to the compound of formula 1 via the lactam compound of the formula 10 (Scheme 3). In this instance, compound of formula 6 (e.g., wherein R1=ethyl) is treated with a base metal catalyst under neutral conditions to reduce the nitrile moiety to an amino moiety. Base metal catalysts effective for the nitrile reduction include nickel-, cobalt-, or copper-aluminum alloy catalysts. A preferred catalyst is a cobalt-aluminum alloy catalyst such as that sold as Raney cobalt catalyst by Engelhard Corporation. Suitable solvents for the reduction include ethanol, methyl t-butyl ether, tetrahydrofuran, isopropanol, and the like. The lactam is hydrolyzed under basic conditions such as 10 N hydroxide solution or under acidic conditions using a suitably strong acid such as 12 N HCl. Preferably the lactam is hydrolyzed under acidic conditions using a mineral acid. DFMO is conveniently isolated as its monohydrochloride monohydrate salt as described above. 
In Process C, the nitrile moiety of the compound of the formula 5 (preferably wherein R2 is aryl) is reduced to an amine before the 2-amino protecting group is removed (Scheme 4). Compound of the formula 5 is treated with a base metal catalyst to reduce the nitrile moiety and provide the compound of the formula 11. Base metal catalysts that can be used for this reduction reaction include nickel-, cobalt-, or copper-aluminum alloy catalysts. Preferably the catalyst is a cobalt-aluminum alloy catalyst. Solvents useful in the reduction reaction include alcohols, e.g., ethanol and ethers, e.g., methyl t-butyl ether. The Schiff""s base can be removed by acid hydrolysis, as described above for Process A, and the ester group is further removed to complete the preparation of DFMO. 
In Process D, the amino protecting group in the compound of formula 5, is switched from a Schiff""s base protecting group to an amide (or carbamate) protecting group (Scheme 5). The compound of the formula 8, wherein R4 is linear or branched C1 to C4 alkyl, alkoxy or aryl, is obtained by treating the compound of formula 5 with suitable acylating reagents. The acylating agents include anhydrides, acid chlorides, chloroformates, activated esters, e.g. N-hydroxysuccinimide esters, or other acylating agents well known to those of ordinary skill in the art. Preferably the acylating reagent is acetic anhydride so that R4 is methyl in the compound of formula 8. The acylation reaction can be performed in ethers, dimethylformamide, dimethylacetamide, esters (e.g., ethyl acetate) as well as other solvents, in the presence of an organic base such as triethylamine or pyridine. 
The nitrile group of the compound of formula 8 is then reduced using procedures analogous to those described above for Process A to provide the compound of formula 9. Although the compound of formula 9 can be isolated and further purified, it can be conveniently used in the next step without further purification. In the final step, the ester and amide moieties of the compound of formula 9 are hydrolyzed to provide DFMO. The hydrolysis can be accomplished by first hydrolyzing the carboxylic acid ester moiety with aqueous base followed by acid hydrolysis of the amide moiety with, for example, mineral acids. Alternatively, both the ester and amide moieties are hydrolyzed simultaneously using acidic conditions, e.g., 12 N HCl. DFMO can be isolated and further purified as its monohydrochloride monohydrate salt as described above.
It can be recognized that the compound of formula 1 or its synthetic precursors can be resolved into its individual isomers by resolution techniques well-known to those of ordinary skill in the art. For example, the lactam of the compound of formula can be formed, i.e., the compound of formula 10, and then the acid addition salt of the lactam can be prepared with a homochiral acid such as (+) or (xe2x88x92) binaphthylphosphoric acid as described in U.S. Pat. No. 4,309,442. Other resolving agents, i.e., homochiral acids, well-known in the art could also be employed. Alternatively, chiral reversed phase chromatography techniques can be used to resolve the product if desired.
DFMO is typically produced by the process of the invention as a salt. The salt can be exchanged by a pharmaceutically acceptable salt as needed to provide the desired formulation.
The following examples further illustrate the present invention, but of course, should not be construed as in any way limiting its scope.